Bone materials

ABSTRACT

The invention provides a synthetic bone precursor material comprising amorphous calcium phosphate and a constituent that inhibits the crystallisation of calcium hydroxyapatite to amorphous calcium phosphate. The synthetic bone precursor may comprise a material of formula CaxMgyPO4 wherein Mg acts as an inhibitor for crystallisation. Other crystallisation inhibitors such as pyrophosphate may be present. The inhibitor is capable of dissolving in physiological saline. Loss of inhibitor from the precursor allows crystallisation of hydroxyapatite. The invention relates to use of a synthetic bone precursor to form bone in vivo, by a transformation of the amorphous precursor material to crystalline hydroxyapatite bone material by the leaching action on inhibitory ions body fluids. it is envisaged that the material will be useful in facilitating bone repair, inducing bone formation or assisting in the attachment of prostheses.

This application is a 371 of PCT/GB93/01519, filed on Jul. 20, 1993.

BACKGROUND OF THE INVENTION

The present invention relates to materials useful in producing bonemineral or similar material; to their preparation; and to their use.

Bone has organic and inorganic constituents. Most of the strength is dueto the inorganic material, of which the major part is crystallinecalcium phosphate, particularly hydroxyapatite, Ca₅ (OH) (PO₄)₃. Thiscrystalline material is strong, stable and essentially insoluble.

SUMMARY OF THE INVENTION

We have now found certain substantially amorphous materials that can betransformed under controlled conditions into crystalline bone materialwhich may be of natural type or similar strong, stable material. Thus inone aspect the invention concerns use of certain precursor materials inthe manufacture of a composition for use in a therapeutic technique inwhich bone is built up in vivo by transformation of the composition toproduce bone material.

The precursor materials contain, as principal inorganic constituents,amorphous non-stoichiometric compounds containing calcium and phosphateions. The phosphate ions may be mainly or wholly derived from phosphoricacid (H₃ PO₄); they may include a proportion of ions derived frompyrophosphoric acid (H₄ P₂ O₇). Generally the compounds containmagnesium ions.

The precursor materials may be essentially inorganic, particularly whenmade synthetically. Alternatively they may include organic components.These can be deliberately included in synthetic materials, or benaturally present in naturally-derived materials.

Generally, the number of moles of magnesium Der mole of calcium is inthe range 0.001 to 1; for example 0.01 to 0.40 and preferably 0.1 to0.16

Material may be synthesised by bringing together calcium ions, magnesiumions, and phosphate ions in a buffer solution under conditions-such thata material of desired composition is precipitated. Other components canbe included in the reaction solution if desired.

Naturally-derived materials can be prepared from naturally-occurringdeposits of amorphous calcium/magnesium/phosphate minerals. Suitabledeposits occur in the soft tissue of many invertebrates (see. e.g. G. L.Becker et al., J. Cell Biology, (1974), 61, 316-326 at page 322 andreferences cited therein), notably the intracellular granules from thedigestive glands of various invertebrates, particularly crabs and snailse.c. as described in K. Simkiss et al, j.Inora.Biochem, (1990), 39,17-23, and G. L. Becker et al, op cit. Thus as described by Simkiss etal, granules can be extracted from C.maenas by homogenisation of thedigestive gland and centrifugation in water to afford a clean whiteproduct. This product consists largely of inorganic phosphate, with themetal ions being mainly calcium but with some magnesium (the molar ratiobeing about 9: 1). The phosphate includes ions derived from phosphoricand pyrophosphoric acids, primarily from phosphoric acid. There are alsoorganic components, mainly adenosine phosphates. (In general, suchgranule-derived material may contain significant amounts of nucleosidesand glycoproteins.)

Materials may be prepared or selected which contain differentproportions of one or more crystallisation inhibitor components such asmagnesium and/or pyrophosphate and/or organic components, in order tocontrol the rate of conversion to bone material under predetermined(generally physiological) conditions. At its simplest, a precursormaterial is an amorphous composition which contains calcium andphosphate ions as primary constituents together with inhibitorcomponents (notably magnesium and/or pyrochosphate ions) which inhibitits transformation to a crystalline form (generally based onhydroxyapatite). In use the precursor material gradually loses inhibitorcomponents (in particularly, magnesium ions) by leaching action toambient body fluids, and undergoes transformation into crystallinehydroxyapatite. Where pyrophosphate ions are used as crystallisationinhibitor the molar ratio of P₂ O₇ to PO₄ is in the range 0.001 to 0.2,preferably 0.01 to 0.1. Other inhibitor components which could be usedin the invention are citrate ions and/or acrylate ions.

In vivo, the precursor material is applied to a site where bone growthis required. The inhibitors will gradually be lost to body fluid,leading to gradual transformation to bone mineral.

Because the lixiviated mineral is a biologically compatible product itis unlikely to produce any allergic effects. Furthermore thetransformation of amorphous material into bone mineral is slow, e.g.taking days, so that it is likely to integrate into normal healingprocesses. The bone mineral that is formed is believed to be compatiblewith collagen mineralisation which would make it particularly useful forassisting in bone repair. It is envisaged that the material will beuseful in facilitating bone repair, inducing bone formation or assistingin the attachment of prostheses.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows in trace A, an X-ray diffraction pattern ofnaturally-derived material prepared as specified in Example (1) below,and shows in trace B, an X-ray diffraction pattern of the same materialafter having been placed in physiological saline solution, inhibitorions leached, and the sample isolated, washed and dried.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Some examples will now be described in more detail with reference to theaccompanying drawing in which: FIG. 1 shows X-ray diffraction data foramorphous material and crystalline material.

EXAMPLES

(1) Precursor Materials: Naturally Derived

Material was isolated from C.maenas and characterised as described inSimkiss et al, op.cit, the content of which is incorporated herein byreference.

(2) Precursor Materials: Synthetic

Material was prepared by techniques adapted from Termine, J. D. et al,Arch. Biochem.Biphys., (1970), 140, 307-317 and 318-325, the content ofwhich is incorporated herein by reference.

Calcium chloride solutions were made up in buffer (Tris HCl) to maintainthe pH at about 7.4. Magnesium chloride was added to these solutions toproduce a range of magnesium concentrations, the Mg: Ca mole ratioranging from 1:1 to 0.01:1. A buffered phosphate solution (based onphosphoric acid) was added. The total concentrations were selected toapproximate to `natural` conditions: ion strength ca 0.15 (and pH ca7.4). The solutions were stirred and the resulting precipitates werecollected by centrifugation, followed by washing and drying at 60° C.The products were characterised by elemental analysis, IR spectroscopyand X-ray diffraction.

Transformation

FIG. 1 shows in trace A, an X-ray diffraction pattern ofnaturally-derived material prepared as specified at (1). A sample of thematerial was place, in physiological saline solution. The inhibitor ionswill leach from the material. After many hours the sample was isolated,washed and dried and its X-ray diffraction pattern was determined again.This is trace B. It can be seen that there is clear evidence ofordering. That is, the material is no longer amorphous. The peaksrepresenting d-spacings of crystalline hydroxyapatite (3.42 Å and 2.80Å) are indicated.

Similar experiments were carried out with the synthetic samples preparedas specified at (2). It was found that the time taken to effecttransformation varied with the concentration of magnesium in the sample.Samples in which some phosphate was substituted by pyrophosphate showeda similar variation in transformation time with pyrophospateconcentration.

The rationale behind this reaction probably involves the following:

(i) Because the minerals are. amorphous they are more soluble than anequivalent crystal. They therefore start to dissolve.

(ii) As the minerals dissolve they tend to lose their high Mg content tothe saline (in vivo, to the body fluids). This removes the inhibition tothe nucleation of hydroxyapatite.

(iii) Bone mineral, which is less soluble than the amorphous deposit,now begins to form.

(iv) It is thought that initial bone deposits normally occur in gapregions of the organic collagen fibres. The crystals of bone in thesecollagen gaps are generally smaller than normal, probably because theycontain magnesium ions. The bone crystals induced by the amorphousdeposits may be of a similar type depending upon how fast the magnesiumis lost.

(v) Because the bone mineral that has formed from these amorphousdeposits is similar to normal bone it should integrate into the normalbone structures of the body and even be capable of being remodelled invivo. In this it differs fundamentally from artificial adhesives.

It is possible to provide a wide range of materials based on amorphousCa_(x) MG_(y) PO₄, with different x:y ratios. These will have differentsolubilities and rates of transformation into bone mineral. This willinfluence their speed of cementing and the properties of the mineralinduced. Thus it is possible to produce a series of simple amorphousmaterials which

(i) transform into bone mineral at predetermined rates (possibly withvarying adhesion properties) when exposed to the body fluids

(ii) produce bone minerals of different physical properties (e.g.crystallinity)

(iii) integrate into normal bone structures

(iv) are capable of being remodelled in the body and

(v) may act as a booster mechanism for normal bone repair.

Such substances are of value in providing an interface to assist theattachment of protheses, promoting normal bone repair and replacingnormal mineral in damaged bones or even teeth (and the term "bonematerial" as used herein is to be understood to cover synthetic toothmaterial).

The amorphous compositions of the invention could also be included in amatrix containing other materials: for example they could be integratedin a matrix with a biologically active moeity to provide an "activecomposite".

Furthermore, according to this invention it is possible to mix severalamorphous calcium phosphate materials chosen to have different rates oftransformation so as to provide for a series of transformations (eitheroverlapping in time or sequential). Transformation time can becontrolled by choice of inhibitor (i.e. one or several ions which may bethe same or different for each amorphous calcium phosphate constituentof the mixture) and choice of inhibitor concentration and/or solubility.

An important aspect of the invention resides in the biomedical useswhich may be made of the materials. A slow transformation with a varyingrange of the inhibiting ions (e.g. magnesium of pyrophosphate)encourages natural bone formation and repair mechanisms.

A mixture of fast-setting and slow-setting compositions of the inventionwill encourage vascularisation of an adhesive material such as a polymethyl methacrylate cement and made such mixtures suitable for coatingthe contact area of implanted bone prostheses.

When manipulated into pre-formed shapes, mixtures of fast andslow-setting materials may be used in spinal surgery as bone-graftingmaterials and bone filling materials for dealing with the collapse ofintervertebral discs. No artificial material has yet been found suitablefor this application which has had to resort to the use of donor bonematerial from the pelvis of the same patient.

We claim:
 1. A method in which synthetic bone material is formed in vivawhich comprises:positioning an amorphous precursor in the body at thepoint where the formation of bone is desired, said precursor consistingessentially of a solid mixture of:a) at least one amorphous calciumphosphate constituent, with the exception of tetra calcium phosphate(Ca₄ (PO₄)₂)O), and; b) at least one of magnesium ion and pyrophosphateion constituent which acts as an inhibitor for crystallisation ofhydroxyapatite (Ca₅ (OH)(PO₄)₃ from amorphous calcium phosphate untilthe solid mixture is placed in a body where bone growth is required andcomes in contact with body fluids, which inhibitor is capable ofdissolving in physiological saline and is capable of leaching from theprecursor to cause crystallisation of hydroxyapatite therefrom; andtransforming the amorphous precursor into crystalline bone material bythe effect of the leaching action of ambient body fluids on inhibitor b.2. A method of treatment which results in the formation of syntheticbone material in vivo, said method comprising applying an effectiveamount of an amorphous precursor to the body of a human or animalpatient at the point where bone material is to be formed, said precursorconsisting essentially of a solid mixture of:a) at least one amorphouscalcium phosphate constituent, with the exception of tetra calciumphosphate (Ca₄ (PO₄)₂)O), and; b) at least one of magnesium ion andpyrophosphate ion constituent which acts as an inhibitor forcrystallisation of hydroxyapatite (Ca₅ (OH)(PO₄)₃ from growth isrequired and comes in contact with body fluids,which inhibitor iscapable of dissolving in physiological saline and is capable of leachingfrom the precursor to cause crystallisation of hydroxyapatite therefrom;and treating said human or animal patient by transforming the amorphousprecursor into crystalline bone material by the effect of the leachingaction of ambient body fluids on inhibitor b.